Erythropoietin (EPO) is a glycoprotein hormone with 165 amino acids, 4 glycosylation sites on amino-acid positions 24, 38, 83, and 126, and a molecular weight of about 34,000. It is initially produced as a precursor protein with a signal peptide of 23 amino acids. EPO can occur in three forms: α, β, and asialo. The α and β forms differ slightly in the carbohydrate components, but have the same potency, biological activity, and molecular weight. The asialo form is an α or β form with the terminal carbohydrate (sialic acid) removed. The DNA sequences encoding EPO have been reported. See, Lin (1987) U.S. Pat. No. 4,703,008.
EPO stimulates mitotic division and the differentiation of erythrocyte precursor cells and thus ensures the production of erythrocytes. It is produced in the kidney when hypoxic conditions prevail. During EPO-induced differentiation of erythrocyte precursor cells, there is induction of globin synthesis and increases in the synthesis of the heme complex and in the number of ferritin receptors. This makes it possible for the cell to take on more iron and synthesize functional hemoglobin. Hemoglobin in mature erythrocytes binds oxygen. Thus, the erythrocytes and the hemoglobin contained in them play a key part in supplying the body with oxygen. The complex processes which have been described are initiated by the interaction of EPO with an appropriate receptor on the cell surface of the erythrocyte precursor cells. See, e.g., Graber and Krantz (1978) Ann. Rev. Med. 29:51-66.
EPO is present in very low concentrations in plasma when the body is in a healthy state wherein tissues receive sufficient oxygenation from the existing number of erythrocytes. This normal low concentration is enough to stimulate replacement of red blood cells which are lost normally through aging.
The amount of EPO in the circulation is increased under conditions of hypoxia when oxygen transport by blood cells in the circulation is reduced. Hypoxia may be caused by loss of large amounts of blood through hemorrhage, destruction of red blood cells by over-exposure to radiation, reduction in oxygen intake due to high altitudes or prolonged unconsciousness, or various forms of anemia. In response to tissues undergoing hypoxic stress, EPO will increase red blood cell production by stimulation of proliferation of erythroid progenitor cells. When the number of red blood cells in circulation is greater than needed for normal tissue oxygen requirements, EPO in circulation is decreased.
Because EPO is essential in the process of red blood cell formation, the hormone has potentially useful applications in both the diagnosis and the treatment of blood disorders characterized by low or defective red blood cell production. Recent studies have provided a basis for the projection of efficacy of EPO therapy in a variety of disease states, disorders, and states of hematologic irregularity, including: beta-thalassemia (see, Vedovato et al. (1984) Acta. Haematol. 71:211-213); cystic fibrosis (see Vichinsky et al. (1984) J. Pediatric. 105:15-21; pregnancy and menstrual disorders (see Cotes et al. (1993) Brit. J. Obstet. Gyneacol. 90:304-311; early anemia of prematurity (see Haga et al. (1983) Acta Pediatr. Scand. 72:827-831); spinal cord injury (see Claus-Walker et al. (1984) Arch. Phys. Med. Rehabil. 65:370-374); space flight (see Dunn et al. (1984) Eur. J. Appl. Physiol. 52:178-182); acute blood loss (see Miller et al. (1982) Brit. J. Haematol. 52:545-590); aging (see Udupa et al. (1984) J. Lab. Clin. Med. 103:574-580 and 581-588 and Lipschitz et al. (1983) Blood 63:502-509; various neoplastic disease states accompanied by abnormal erythropoiesis (see Dainiak et al. (1983) Cancer 5:1101-1106 and Schwartz et al. (1983) Otolaryngol. 109:269-272); and renal insufficiency (see Eschbach et al. (1987) N. Eng. J. Med. 316:73-78).
Purified, homogeneous EPO has been characterized. See Hewick, U.S. Pat. No. 4,677,195. A DNA sequence encoding EPO was purified, cloned and expressed to produce synthetic polypeptides with the same biochemical and immunological properties. A recombinant EPO molecule with oligosaccharides identical to those on the natural material has also been produced. See, Sasaki et al. (1987) J. Biol. Chem. 262:12059-12076.
Despite the availability of purified recombinant EPO, little is known concerning the mechanism of EPO-induced erythroblast proliferation and differentiation. The specific interaction of EPO with progenitors of immature red blood cells, platelets, and megakaryocytes remains to be characterized. This is due, at least in part, to the small number of surface EPO receptor molecules on normal erythroblasts and on the erythroleukemia cell line. See, Krantz and Goldwasser (1984) Proc. Natl. Acad. Sci. USA 81:7574-7578; Branch et al. (1987) Blood 69:1782-1785; Mayeux et al. (1987) FEBS Letters 211:229-233; Mufson and Gesner (1987) Blood 69:1485-1490; Sakaguchi et al. (1987) Biochem. Biophys. Res. Commun. 146:7-12; Sawyer et al. (1987) Proc. Natl. Acad. Sci. USA 84:3690-3694; Sawyer et al. (1987) J. Biol. Chem. 262:5554-5562; and Todokoro et al. (1988) Proc. Natl. Acad. Sci. USA 84:4126-4130.
Cross-linked complexes between radioiodinated EPO and cell surface proteins suggest that the cell surface proteins comprise two polypeptides having approximate molecular weights of 85,000 daltons and 100,000 daltons, respectively. More recently, the two cross-linked complexes have been subjected to V8 protease digestion and have been found to have identical peptide fragments, suggesting that the two EPO-binding polypeptides may be products of the same or very similar genes. See, Sawyer et al. (1988) supra. Most cell surface binding studies, however, have revealed a single class of binding sites, averaging 300 to 600 per cell surface, with a Kd of approximately 800 pM (picomolar). See, Sawyer et al. (1987) Proc. Natl. Acad. Sci. USA 84:3690-3694. However, EPO-responsive splenic erythroblasts, prepared from mice injected with the anemic strain (FVA) of the Friend leukemia virus, demonstrate a high and a low affinity binding site with dissociation constants of 100 pM and 800 pM, respectively. See, Sawyer et al. (1987) J. Biol. Chem. 262:5554-5562 and Landschulz (1989) Blood 73:1476-1478. The DNA sequences and encoded peptide sequences for murine and human EPO receptor proteins have been described. See, D'Andrea et al., PCT Patent Publication No. WO 90/08822 (published 1990).
The availability of cloned genes for the EPO receptor (EPO-R) facilitates the search for agonists and antagonists of this important receptor. The availability of the recombinant receptor protein allows the study of receptor-ligand interaction in a variety of random and semi-random peptide diversity generation systems. These systems include the “peptides on plasmids” system described in U.S. patent application Ser. No. 778,233, filed Oct. 16, 1991, issued as U.S. Pat. No. 5,270,170, the “peptides on phage” system described in U.S. patent application Ser. No. 718,577, issued as U.S. Pat. No. 5,432,018 and in Cwirla et al., August 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382, the “encoded synthetic library” (ESL) system described in U.S. patent application Ser. No. 946,239, filed Sep. 16, 1992, which is a continuation-in-part application of Ser. No. 762,522, filed Sep. 18, 1991, now abandoned, the “very large scale immobilized polymer synthesis” system described in U.S. patent application Ser. No. 492,462, filed Mar. 7, 1990, now U.S. Pat. No. 5,143,854, and those systems described in PCT patent publication No. 90/15070, published Dec. 13, 1990, U.S. patent application Ser. No. 624,120, filed Dec. 6, 1990, now abandoned, Fodor et al., 15 Feb. 1991, Science 251:767-773, Dower and Fodor, 1991, Ann. Rep. Med. Chem. 26:271-180, and U.S. patent application Ser. No. 805,727, filed Dec. 6, 1991, now U.S. Pat. No. 5,424,186.
Novel peptides that interact with the EPO-R have been discovered, and have been disclosed in U.S. Pat. No. 5,773,569 to Wrighton et al. The Wrighton et al. peptides are generally single polypeptide chains about 10 to 40 amino acids in length, and contain a pair of cysteine residues separated by eight amino acids. These EPO-R-binding peptides were found to bind to or otherwise interact with the EPO-R, and were thus presumed to be useful for studying the biological activities of the EPO-R and for treatment of disease involving a deficiency of EPO. A related patent, U.S. Pat. No. 5,830,851 to Wrighton et al., pertains more specifically to therapeutic applications of the aforementioned peptides, and focuses on the use of the compounds to treat disorders such as end-stage renal failure or dialysis, anemia associated with AIDS, autoimmune diseases, malignancies, and chronic inflammatory disease, beta-thalassemia, cystic fibrosis, spinal cord injury, acute blood loss, aging, and neoplastic disease states accompanied by abnormal erythropoiesis.
There remains a need, however, for highly active compounds that bind very specifically to the EPO-R, both for studies of the important biological activities mediated by the receptor and for treatment of diseases, disorders and conditions associated with an EPO deficiency. There remains a further need for improved synthetic methods for the production of such compounds. The present invention provides such compounds and methods, and also provides pharmaceutical compositions and methods for using the compounds as therapeutic agents.